2.3. Interaction of Endothelial Cells on 3D Matrices
This protocol is extracted from research article:
Assessment of Electrospun Pellethane-Based Scaffolds for Vascular Tissue Engineering
Materials (Basel), Jul 1, 2021; DOI: 10.3390/ma14133678

The scaffolds (discs with a diameter of 10 mm) were pre-incubated with the culture medium for 2 h to achieve complete moistening of the material. Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical vein of newborn by collagenase IV. Culture medium was removed from the wells before seeding the HUVEC cells on the scaffolds (2000–8000 cells per well). The seeding density of cells was about 50–65% of the monolayer. Control cells were cultivated in parallel on tissue culture polystyrene. The viability of endothelial cells was assessed after 48-h cultivation on the surface of the matrices. The culture medium was removed and the cells were incubated with Alamar Blue® (Invitrogen, Eugene, OR, USA), as earlier described [26]. Matrices without cells were used as a control for assessing the dye sorption on the tested materials.

The matrices with HUVEC after 48 h incubation were washed twice with phosphate buffer to remove non-adherent cells and fixed in 2% formaldehyde in a phosphate buffer for 30 min. Graded ethanol series (50%, 70%, 90%, and 100%) were used for sample dehydration before incubation in a mixture of hexamethyldisilazane (HMDS) with ethanol (1:1), and then 100% HMDS in order to prepare 3D matrices with cells for study by a EVO 10 (Carl Zeiss AG, Jena, Germany) scanning electron microscope.

HUVEC cells cultivated at 3D matrices during 48 h were fixed in 2% formaldehyde in a phosphate buffer and stained with Hoechst 33342 (Invitrogen, USA) and Phalloidin-TRITC (Sigma, USA) for 20 min at 37 °C for subsequent fluorescent microscopy. For this study, we used a confocal laser scanning microscope LSM 780 NLO (Carl Zeiss, Jena, Germany) at 405 nm to detect cell nuclei and at 543 nm to detect actin microfilaments with ZEN 2 software (Carl Zeiss Inc., München, Germany).

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